(1) Nucleotide analogs have been introduced as structural probes into active sites of dodecameric glutamine synthetase from E. coli. Various analogs of ATP that are substituted at the 6- or 8-position of the purine ring have been further modified with spectrophotometric and fluorometric probes or an electron dense Pt(II) marker. Fluorescence energy transfer measurements were made with enzyme derivatives containing 1 or 2 eq of fluorescent nucleotide donor per dedecamer and ADP or acceptor analogs at the remaining active sites. Energy transfer measurements made last year showed that active site probes in the dodecamer are widely separated (56-60 Angstrom). Using the same fluorescent derivatives, zinc-induced face-to-face stacking of enzyme dodecamers had a second-order rate constant of 50000/M.s at 25 degrees C and fluorescent donor and acceptor probes in layered dodecamers were found to be about 36 Angstrom apart. The zinc-induced stacking of enzyme dodecamers also is being studied by calorimetry at 22.5, 30.0, and 38.0 degrees C in order to investigate the forces governing macromolecular assembly reactions. Polymerization rates increase with increasing temperatures with an Arrhenius activation energy of 17.7 kcal/mol. A value of DeltaCp = -850 cal/K.mol has been estimated for polymerization. (2) Glutamine synthetase from S. typhimurium has been labeled at active sites or adenylylation sites with mercapto nucleotide.platinum(II) and sent to UCLA for crystallization and X-ray analysis. (3) Bovine brain glutamine synthetase has been found to have two essential divalent cation sites/subunit -- a structural site and a higher affinity nucleotide-metal ion site which is filled after the first site is occupied by Mn(II) or Mg(II). Although the enzyme is active with either Mg(II) or Mn(II) in vitro, only Mg(II) is bound to the brain enzyme in vivo. An allosteric site for chloride, L-glutamate, or arsenate is indicated. (4) A reversible thermal transition of dodecameric glutamine synthetase from E. coli was found to involve a melting of active site structures. (5) Studies of Zn(II) binding to regulatory proteins are in progress.
